J . Med. Chem. 1986,29, 778-783
778
1.7 (d, J = 7.5,3 H), 5.0 (dd, J = 7.5, 1.75, 1 H), 6.2 (d, J = 1.75, 1 H), 6.4 (s, l H ) , 7.2 (m, 4 H); anal. (Cl2HloNO4C1)C, H , N]. Compound 84 [0.218 g, 4%; m p 114-116 “C; IR (KBr) 1817,1723; NMR (CDCl,) 4.5 (d, J = 5 , 2 H), 5.3 (m, 2 H), 6.0 (m, 1 H), 6.4 (5, 1 HI, 7.1 (m, 4 H); anal. (C,,H,,,NO,Cl) C, H , N1. 5 4 2-Chloro-6-[ (cyc1opropyimethyi)oxy ]phenyl]oxazolidine-2,4-dione (83). A solution of compound 57 (2.00 g, 8.71 mmol), potassium tert-butoxide (1.95 g, 17.42 mmol), cyclopropylmethyl alcohol (10 mL), and 10 mL of MezSO was heated a t reflux for 3 h, cooled, and poured into 200 mL of 1 N HCl. The aqueous was extracted with-three portions of ethyl acetate, and
the pooled organic layers were washed with water and brine, dried with MgSO.,, filtered, and concentrated in vacuo to a brown oily solid, which recrystallized from ethyl acetate/hexane [1.37 g, 65%; mp 188-189 O C ; IR (KBr) 1827,1744; anal. (C13H12N04C1)C, H,
N1.
Acknowledgment. We gratefully acknowledge Paul R. Kelbaugh, Derek Tickner, DeCusatis and Harry R. h ~ ~ a for r dtheir chemical contributions and Dwight MacDonald, Weldon Horner, and Kart Grizzuti for their biological expertise.
Crystal Structures of a- and P-Funaltrexamine: Conformational Requirement of the Fumaramate Moiety in the Irreversible Blockage of p Opioid Receptors Jane F. Griffin,*+Dennis L. Larson,t and Philip S. Portogheset Molecular Biophysics Department, Medical Foundation of B u f f a l o , B u f f a l o ,N e w York 14203, and Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455. Received M a y 13, 1985 CY- and /3-funaltrexamine (m-and /3-FNA, la and l b ) are naltrexone derivatives differing only in chirality a t C(6). Both epimers bind t o fi opioid receptors in G P I and MVD preparations, but only the @-epimerirreversibly blocks these receptors in both preparations. In an effort to investigate the reasons for this difference, we have determined the molecular structures of la and l b by X-ray diffraction techniques. T h e two epimers have almost identical conformations in the fused ring system except for ring C, which is observed in a twist-boat conformation in n-FNA and a chair in /3-FNA. As a result the electrophilic fumaramate moieties are equatorial in both structures and orthogonal to one another when the fused rings are superimposed. In the crystal structure of @-FNAthere is a close intermolecular contact between a phenolic oxygen and the fumaramate double bond that can serve as a model for nucleophilic attack on the fumaramate group. When l a and l b are superimposed, the fumaramate double bond of l a is more than 2 A away from that in its epimer l b and in the wrong orientation for nucleophilic attack from the proposed direction to take place. T h e results of this study are consistent with a model that postulates the involvement of two consecutive recognition steps leading to the irreversible blockage by /3-FNA (Sayre, L. M.; Larson, D. L.; Fries, D. S.; Takemori, A. E.; Portoghese, P. S. J. Med. Chem. 1983,26,1229) and underscores the importance of the second recognition step in conferring selectivity in the Michael addition of a nucleophile to the fumaramate group.
0-Funaltrexamine (P-FNA, lb) is a naltrexone-derived nonequilibrium narcotic antagonist that is highly selective for the p-type opioid receptor ~ystem.l-~The available evidence suggests that the nonequilibrium nature of P-FNA arises as a consequence of the reaction of the fumaramate moiety with a putative nucleophile near the recognition locus of the r e ~ e p t o r . ~ - ~
it
”
RLH,R * = N H C O C = C C O ~ C H ~ H
The high selectivity of P-FNA for the p opioid receptor, despite its interaction with other opioid receptor types, has been attributed to the involvement of two consecutive recognition ~ t e p s . ~The - ~ first is reflected by affinity of the ligand for the recognition site; the second involves the proper alignment between the electrophilic center of the ligand with a chemically compatible receptor-based nucleophile. Because two recognition steps rather than one lead to covalent binding, enhanced receptor selectivity (recognition amplification) is obtained. Due to the high selectivity of P-FNA as a nonequilibrium antagonist at p Molecular Biophysics Department.
* Department of Medicinal Chemistry. 0022-2623/86/ 1829-0778$01.50/0
opioid receptors, it has been employed widely as a tool in the investigation of opioid receptor mechanisms.10 In contrast to P-FNA, its epimer a-FNA (la) does not irreversibly block the effects of p receptor agonists but does protect against p-FNA-induced irreversible antag~nism.~ This suggests that both a- and 0-FNA interact with the same site, but the second recognition step is achieved only with 0-FNA. Since there is no substantial difference between the reactivity of la and l b in s o l ~ t i o nan , ~obvious explanation for the observed difference in irreversible antagonism between these epimers may be related to (1) Portoghese, P. S.; Larson, D. L.; Sayre, L. M.; Fries, D. S.; Takemori, A. E. J . Med. Chem. 1980, 23, 233. (2) Takemori, A. E.; Larson, D. L.; Portoghese, P. S. Eur. J . Pharmacol. 1981, 70, 445.
(3) Ward, S. J.; Portoghese, P. S.; Takemori, A. E. J . Pharmacol. E x p . T h e r . 1982, 220, 494. (4) Ward, S. J.; Portoghese, P. S.; Takemori, A. E. Eur. J . Pharmacol. 1982, 80, 377. ( 5 ) Ward, S. J.; Portoahese. P. S.; Takemori, A. E. E m . J . Pharm a d . 1982,85, 163. (6) Portoehese. P. S.: Takemori. A. E. In “The Chemical Reeulation 07 Biological’Mechanisms”;Creighton, A. M., Turner, S., Eds.; The Royal Society of Chemistry: London, 1982; p 181. (7) Portoghese, P. S.; Takemori, A. E. In “Natural Products and Drugs Development”; Kofod, H., Ed.; Munksgaard: Copenhagen, Denmark, 1984; p 421. (8) Sayre, L. M.; Larson, D. L.; Fries, D. S.; Takemori, A. E.; Portoghese, P. S. J . M e d . Chem. 1984, 27, 1325. (9) Sayre, L. M.; Larson, D. L.; Fries, D. S.; Takemori, A. E.; Portoghese, P. S. J . Med. C h e m . 1983. 26, 1229. (10) Takemori, A. E.; Portoghese, P. S.Ann. Reu. Pharmacol. 1985, 25, 193.
0 1986 American Chemical Society
Journal of Medicinal Chemistry, 1986, Vol. 29, No. 5 779
Crystal Structures of a- a n d p-Funaltrexamine
0
Q
n
Figure 1. Stereo ORTEP views of a-funaltrexamine(top) and P-funaltrexamine(bottom). The C-ring atoms are stippled to highlight the ring conformations: a twist-boat in a-FNA and a chair in 0-FNA.
different orientations of the electrophilic centers in these molecules. In order to provide experimental evidence for the preferred conformation of the fumaramate group, we have determined by X-ray diffraction studies the molecular structures of both a- and P-FNA.'l X-ray Results Stereo ORmP views Of the crystallographically Observed structures Of la and lb are shown in Figure The two epimers have almost identical conformations in the fused ring moiety except for ring C: in a-FNA ring C is observed in a twist-boat conformation and in @-FNAring C is in a chair conformation (see I)' The ring Of dihydromorphine analogues has always been observed in a flattened or distorted chair in previous crystallographic studies.12-17 The torsion angles for the C ring of naloxone12 (11) Griffin, J. F.;Portoghese, P. s. Abstract 03.1-15, XIIIth Congress of the IUCr, Hamburg, Germany, Aug 1984. (12) Karle, I. L. Acta Crystallogr., Sect. B 1974, B30, 1682. (13) Sime, R. J.; Dobler, M.; Sime, R. L. Acta Crystallogr., Sect. B 1976, B32, 2937. (14) Kalman, A.; Ignath, Z.; Simon, K.; Bognar, R.; Makleit, S. Acta Crystallogr., Sect. B 1976, B32, 2667. (15) Sasvari, K.; Simon, K.; Bognar, R.; Makleit, S. Acta Crystallogr., Sect. B 1974, B30, 634. (16) Sime, R. J.; Dobler, M.; Sime, R. L. Acta Crystallogr., Sect. B 1976, B32, 809. (17) Iijima, I.; Rice, K. C.; Silverton, J. V. Heterocycles 1977, 6, 1157.
are given in Table I for comparison. The C ring conformations in a- and 6-FNA result in the fuamaramate chain on C6 being equatorial to ring C in both compounds. The side chains on N(1) differ in conformation: the cyclopropyl ring is trans to C(16) (see atomic numbering scheme on figure in Table I) and on the OH(14) side of the piperidyl ring in P-FNA and trans to C9 and approximately in the plane of the piperidyl ring on the C(15)C(16) side in a-FNA. These confomations have both been observed previously, the former in cyclazocinelE and the latter in gemazocine.lg The epimeric funaltrexamines l a and lb differ in hydrogen-bonding patterns even though they crystallize in the same space group with one water molecule and a halide ion per asymmetric unit. The hydrogen-bond geometry is given in Table There is no hydrogen bond involving O(21) in a-FNA while all donors and acceptors are involved in hydrogen bonds with good geometryin P-FNA. The fumaramate groups are approximately planar in both structures with the groups at c(21)and C(24) cis to the double bond. The ester oxygen is rotated slightly out of the plane in 0-FNA. The torsion angles describing the fumaramate groups are listed in Table IB. (18) Karle, I. L.; Gilardi, R. D.; Fratini, A. V.; Karle, J. Acta Crystallogr., Sect. B 1969, B25, 1469. (19) Gelders, Y. G.; DeRanter, C. J.; Schenk, H. Acta Crystallogr., Sect. B 1979, 835, 699.
780 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 5
Griffin et al.
Table I. Observed Conformational Geometry from
Crystallographic Studies
A. Conformation of Ring C wFNA, torsion angle deg 8-FNA, deg C(13)-C(5)-C(6)- -41.2 42.6
--__
C(7)
C(5)-C(6)-C(7)C(8)
C(6)-C(7)-C(8)-
naloxone,” deg 31.0
58.9
-59.2
-47.5
-19.3
65.3
60.6
-33.1
-55.2
-58.8
51.0
44.2
43.6
-12.8
-36.7
-28.8
36.0 AC,(6) = 23.5‘
50.5 AC,(5) = 10.gd
45.0 AC,(6) = 22.5
AC’,(7-83
AC,(6) = 20.1
AC,(7) = 19.7
= 19.5 ACz(5) =
ACZ(5-6) = 21.8 ACz(5-6) = 17.9
18.8 ACz(6-7)
ACZ(6-7) = 20.7 AC,(6-7) = 29.8
c(14)
C(7)-C(8)-C(14)C(13) C(8)-C( 14)C ( 13)-C( 5 ) C(14)-C( 13)-
i
av torsion angle asymmetry parametersb
f
\
C(5)-C(6)
= 21.1
conformation
twist-boat distorted chair
flattened chair
B. Fumaramate Side-Chain Conformation torsion angle a-FNA, deg P-FNA, deg H(6)-C(6)-N(6)-C(21) -16.1 12.4 C(5)-C(6)-N(6)-$(21) -134.1 128.4 C(7)-C(6)-N(6)-C(21) 101.0 -107.3 C(5)-C(6)-N(6)-H(6N) 44.4 -49.9 C(7)-C(6)-N(6)-H(6N) -80.5 74.4 C(S)-N(S)-C(2l)-C(22) 179.0 -178.3 C(6)-N(S)-C(21)-0(21) 0.6 1.1 N(6)-C(Zl)-C(22)-C(23) -167.9 -163.1 0(21)-C(2l)-C(22)-C(23) 10.4 17.5 C(2l)-C(22)-C(23)-C(24) 177.3 172.8 C(22)-C(23)-C(24)-0(24) -5.8 17.0 C(22)-C(23)-C(24)-0(25) 173.4 -160.5 C(23)-C(24)-0(25)-C(25) -176.3 177.6 C. Hvdroeen-Bond Geometrv donor-acceutor distance. A D-H * A anele. dee a-FNA 2.872 160.2 N(l)-O(W) 168.6 2.835 O(14)-O(24) 3.348 160.3 N(6)-C1 166.8 3.120 0(3)-C1 3.340 158.1 O(W)-CI 3.045 O(W)-014 e 8-FNA 149.1 N(1)-0(21) 2.969 146.7 O (14)-O( W) 2.763 161.1 3.464 N(G)-Br 141.1 3.217 0(3)-Br 146.7 3.034 O(W)-0(24) 164.2 3.239 OW-Br “Reference 12. bDuax, W. L.; Weeks, C. M.; Rohrer, D. C. In “Topics in Stereochemistry”; Allinger, N. L., Eliel, E., Eds.; Wiley: New York, 1976; Vol. 9, pp 280-286. ‘An ideal boat conformation would have AC,(6) = AC,(7-8) = 0.0; an ideal twist would have AC2(5) = ~ W ~ ( 6 - 7=) 0.0. dThe largest asymmetry parameters are given to show distortion. An ideal chair has six asymmetry parameters equal to 0.0. See footnote b above. eHydrogen not located.
6. Figure 2. Stereo views of a least-squares fit of the observed structures of a- (dashed) and 0-FNA (solid). The atoms fit were C l C 5 , C9-C14,03,04,014, N1. Bottom view is made by a 90° rotation about a horizontal axis in the top view. Note how the fumaramate side chains are oriented with respect to the morphine T-shape. The arrow denotes the proposed direction of attack by a receptor nucleophile.
The fumaramate moieties are approximately orthogonal to one another in the two structures. We have made a least-squares fitzoof the two structures, fitting C(l) C(5), C(9) C(14), 0(3), 0(4), 0(14), and N(1) (average intermolecular separation, 0.12 (6) A). A stereo drawing of the superposition is shown in Figure 2. Crystallographic data and refinement parameters are given in Table 11. Atomic coordinates for a-FNa and 6-FNA are listed in Tables I11 and IV, respectively. Spectral Evidence for C-Ring Conformation Crouchz1performed high-field lH NMR on the 6a- and 66-epimers of oxymorphamine (2a and 2b). From J-correlation contour plots and water-eliminated normal spectra for a- and P-oxymorphamine,they showed that in solution ring C exists in a chair conformation in the 66-epimer but is in a twist-boat conformation in the 6a-epimer. This is precisely what is seen in the solid-state conformations of a- and P-FNA. In the latter case, it was easy to explain the observations as due to the fumaramate moiety pref-
-
-
(20) Rohrer, D. C.; Smith, G. D. In “PROPHET Molecules”; Rindone, W., Kush, A., Eds.; Bolt, Beranek and Newman: Cambridge, MA, 1980. (21) Crouch, R. C.; Bhatia, A. V.; Lever, 0. W., Jr. Tetrahedron Lett. 1983, 24, 4801.
Crystal Structures of a - a n d 6-Funaltrexamine
J o u r n a l of Medicinal Chemistry, 1986, Vol. 29, No. 5 781
Table 11. Crystallographic Data wFNA formula formula weight crystal system
P-FNA
C L ~ B N & + , Br-, H2O 554.47 orthorhombic 14.559 (7) A a, A 17.559 (6) b, A 9.413 (8) c, A a, 0, 7 , deg 90.0 2406.(4) A3 volume, A3 1.53 Pcslcdt g/cm3 1156 p , mm-' Mo; X = 0.71069 radiation space group ~12121 m12121 Z 4 4 no. variables 316 316 no. observations 2940 2735 > 3u R factor 0.068 0.102 Rw 0.071 0.079 GOF 1.493 1.870 T,K 298 83 21.82' > 29 < 34.88O 15.250 > 28 < 21.990 25 reflections for cell constant refinement diffractometer Enraf-Nonius CAD4 Fortran P3 0.40 X 0.80 X 0.98 0.04 X 0.12 X 0.98 crystal size, mm crystals grown from aqueous solution aqueous solution structure solved MULTANa/NQESTb MULTAN"/NQEST* weighting scheme experimentalc experimentalC a Main, P.; Lessinger, L.; Woolfson, M. M.; Germain, G.; Declercq, J. P. "MULTAN 77. A system of computer programs for the automatic solution of crystal structures from X-ray diffraction data"; Universities of York, England, and Louvain: Belgium, 1977. DeTitta, G. T.; Edmonds, J. W.; Langs, D. A.; Hauptman, H. Use of negative quartet cosine invariants as a phasing figure of merit: NQEST. Acta Crystallogr., Sect. A 1975, A31, 472. cBlessing, R. H.; DeTitta, G. T. Abstract 06.6-02, XIIth Congress of the IUCr, Ottawa, Ontario, Canada, Aug 1981.
erring the equatorial orientation. It is not as apparent why the amine group prefers the equatorial orientation in both epimers. For this reason we performed MM2pZ2calculations on the epimers of oxymorphamine (2a, 2b). Four structures were generated for input to the MM2p calculations. The twist-boat C-ring conformer of 2a was generated from the crystallographic coordinates of la, substituting an NH, group for the fumaramate in the a-position; the chair C-ring conformer of 2a was generated from the coordinates of lb with an NH2 in the a-position. Similarly the two C-ring conformers of 2b were generated from la and lb with NH2in the @-position.The structures were allowed to refine to their minimum energy. The twist-boat conformer of 2a was calculated to be 1.10 kcal/mol lower in energy than the chair; the chair conformer of 2b was calculated to be 4.11 kcal/mol lower in energy than the twist-boat. That is, the observed conformations are calculated to be lower in energy, but the magnitude of the difference in the a-epimer may be underestimated in light of the spectral evidence in solution. An experimental basis for mapping the reaction coordinate (minimum energy pathway) for the nucleophilic addition to a carbonyl group by nitrogen23and oxygen24 has been determined by examining the geometry of close contacts between the nucleophile and carbonyl carbon in a survey of crystal structures. The authors concluded that the nucleophile approaches along a line, not perpendicular, but forming an angle of -107' with the C=O bond. In the structure of /3-FNA a close intermolecular contact is o b s e r v e d b e t w e e n the C(23) c a r b o n of the fumaramate (22) Allinger, N. L.; Yuh, Y. MMl/MMPl. A program for general
molecular mechanics calculations with the 1973 force field. QCPE No. 400. MM2. Program with more recent force field. QCPE No. 395. MM2p is a version of MM2 containing the T treatment from MMPl amended by D. C. Rohrer (1983). (23) Burgi, H. B.; Dunitz, J. D.; Shefter, E. J.Am. Chem. SOC. 1973, 95,5065. (24) Burgi, H. B.; Dunitz, J. D.; Shefter, E. Acta Crystallogr., Sect. B 1974, B30, 1517.
Czs06NzH31+, Cl-, HzO 510.81 orthorhombic 10.5346 (8) 31.413 (4) 7.5347 (5) 90.0 2493.4 (7) 1.36 1080 Cu; X = 1.5418
Figure 3. Schematic drawing of the proposed p opioid receptor site. The first recognition process, common to la and Ib, involves interactions between the receptor protein and the positively charged nitrogen (-), the phenolic ring (T), the phenolic OH (6-). (G) represents the second recognition site, a nucleophilic residue positioned to attack the fumaramate group of l b but not la.
double bond and the O(3) phenolic oxygen of a neighboring molecule, which appears to distort the fumaramate group. The angle formed by 0(3')-C(23)=C(22) is 118.5O; the distance from O(3') to C(23) of the double bond is 3.19 A. We have included O(3') on the stereo overlap view (with arrows) and propose this as a model for nucleophilic attack on the fumaramate group in the second recognition step leading to covalent binding of the I.L opioid receptor (see Figure 3). The fumaramate group on a-FNA would be positioned approximately orthogonal to the same group in @-FNAand not in an ideal position for nucleophilic attack by the receptor. From the least-squares fit of the fused ring moieties of a- and @-FNA,the C(23) of the a-epimer is more than 2 A away from the C(23) of the @-epimerand appears to be in the wrong orientation for Michael addition to take place (see Figures 2 and 3).
782 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 5 Table 111. Atomic Coordinates (X104) and Equivalent Isotropic Thermal Parameters of a-Funaltrexamine" atom X Y 2 Bi., 4.04 1623 (1) 6240 (1) 169 (2) 5288 (9) 3.73 13428 (6) 328 (2) 5507 (9) 257 (2) 3.69 12154 (6) 11225 (6) 4452 (9) 466 (2) 3.56 11715 (5) 3162 (8) 738 (2) 2.96 1350 (7) 2.83 11867 (5) 1316 (2) 2347 (9) 3.31 1737 (2) 11747 (5) 4151 (9) 3.62 12413 (6) 1727 (2) 4022 (8) 3.14 13827 (6) 1629 (2) 2.80 2550 (8) 15428 (5) 1140 (2) 3.24 15244 (6) 3835 (8) 763 (2) 3.08 609 (2) 3982 (8) 13901 (6) 2.70 2908 (7) 12982 (5) 789 (1) 2.50 1513 (7) 1123 (2) 13198 (5) 1415 (1) 2.61 14231 (5) 2257 (8) 3.04 934 (2) -273 (8) 13642 (5) -155 (8) 3.03 14923 ( 5 ) 710 (2) 17197 (6) 819 (9) 3.86 807 (2) 3.68 716 (2) 17773 (6) -949 (9) -990 (11) 5.56 405 (2) 18847 (7) 291 (2) -1839 (11) 5.32 17639 (8) 2245 (2) 2248 (10) 3.68 9946 (6) 3.66 2522 (9) 2284 (2) 8591 (6) 3.63 1986 (9) 7929 (6) 2622 (2) 2230 (9) 3.62 6577 (6) 2633 (2) 6.17 2099 (13) 4714 (8) 3066 (3) 2.83 15879 (4) 710 (6) 997 (1) 3.71 1850 (1) 10416 (5) 2564 (8) 4.72 404 (1) 4743 (8) 9976 (4) 3.27 1001 (1) 2118 (6) 10983 (3) 904 (5) 3.13 14485 (4) 1730 (1) 4.43 2539 (1) 1724 (7) 10651 (4) 5.07 2778 (8) 5923 (5) 2346 (1) 4.18 1794 (7) 6066 (4) 3015 (1) 7.43 2146 (9) 3414 (2) 1300 (6) 22 4.8 1402 606 122 4 3.9 1602 4.8 7 638 1183 5.9 63 469 969 131 21 3.9 1165 4.4 167 195 1214 4.9 172 283 989 4.7 509 150 1194 4.7 479 201 1224 4.3 487 150 1408 4.3 389 1424 194 3.9 132 312 1610 4.4 483 1554 84 4.4 54 337 1581 4.3 139 1500 190 4.1 -110 117 1374 4.1 73 -80 1301 4.2 -147 63 1524 4.2 61 42 1482 5.0 155 102 1781 152 5.0 1714 50 4.8 -174 1792 93 6.7 -167 45 1954 6.7 6 27 1916 -329 6.5 27 1754 6.5 -128 1699 5 4.8 204 324 816 4.7 133 283 833 7.4 345 300 448 7.4 170 443 338 7.4 125 422 283 H(25C) 7.9 323 H(OW1) 172 348 Estimated standard deviations are given in parentheses. Atomic coordinates for hydrogen atoms are X103. ~~
~~
Other Conformations of the Fumaramate Group The fumaramate group is seen in both a- and 6-FNA structures as a planar moiety with bond distances showing evidence of delocalization through NHCOC=CCO, which is to be expected from a consideration of the chemical
Griffin et al. Table IV. Atomic Coordinates (X104) and Equivalent Isotropic Thermal Parameters of 0-Funaltrexamine" atom X Y 2 B,," 466 (1) 4782 (1) 1.64 6917 (1) 1.52 217 (6) 8030 (6) 3003 (12) 1.74 8568 (7) 181 (6) 4070 (11) 1.42 9085 (6) 4334 (11) 892 (6) 9059 (6) 1636 (6) 1.26 3402 (10) 1.21 2717 (9) 9001 (6) 3106 (6) 0.99 8437 (6) 3468 (7) 3840 (10) 7724 (6) 1.70 3851 (6) 3166 (13) 1.12 7303 (6) 3109 (6) 2261 (10) 1.08 7515 (7) 111 (11) 2048 (7) 7425 (6) 1140 (6) 1.11 868 (11) 8007 (5) 976 (6) 2068 (12) 1.32 8540 (5) 1656 (6) 2283 (9) 0.88 8600 (5) 2558 (6) 1531 (8) 0.73 7828 (6) 2860 (6) 1015 (11) 1.10 9159 (6) 2452 (6) 243 (10) 0.95 1773 (7) 8857 (6) -823 (10) 1.17 1368 (6) 7780 (6) -2387 (11) 1.32 1543 (7) 6964 (6) -2798 (10) 1.34 1526 (7) 6792 (6) -4387 (11) 1.56 2431 (7) -3623 (11) 6793 (7) 1.66 4077 (7) 8847 (6) 1.19 6184 (10) 4832 (6) 9330 (5) 1.50 6800 (12) 5083 (6) 9287 (6) 8157 (13) 1.73 5904 (6) 9720 (6) 1.40 8608 (11) 7081 (6) 2.05 9832 (7) 10310 (11) 2003 (5) -1182 (9) 1.26 8033 (5) 4128 (5) 8842 (5) 1.37 4721 (9) 885 (5) 9604 (4) 1.90 5399 (7) 2433 (4) 9481 (4) 1.46 3482 (7) 3640 (4) 7920 (4) 1.32 82 (8) 8515 (4) 3501 (4) 6874 (8) 1.49 6242 (4) 10238 (4) 1.79 7915 (7) 6254 (4) 9471 (4) 1.81 9797 (8) 1051 (5) 3591 (4) 4446 (9) 2.47 -34 762 289 2.5 2.5 -44 863 469 2.1 366 934 228 451 1.9 290 828 734 400 2.6 409 251 444 787 2.6 289 1.9 251 720 678 186 1.9 338 -25 1.9 225 696 2.4 110 686 131 2.4 750 60 10 924 1.8 312 -26 971 222 63 1.8 920 180 -177 2.0 888 110 -37 2.0 783 -202 2.1 67 2.1 814 -329 146 2.2 652 -210 130 724 -514 136 2.4 2.4 627 -478 122 2.4 626 -344 279 723 2.4 -381 293 970 2.7 608 519 2.4 897 89 1 468 3.0 695 1043 1048 3.0 728 956 1130 976 3.0 761 952 947 4.0 38 589 25 750 4.0 375 3.5 264 -163 806 429 912 3.0 459 3.5 94 516 406 4.0 89 375 382 Estimated standard deviations are given in parentheses. "Atomic coordinates for hydrogen atoms are X103.
constraints in the side chain. This observation combined with the solution and solid-state evidence for the preferred conformation of the C ring (C(6)P-NH2and -fumaramate in a chair conformation and C(6)a-NH2and -fumaramate
Crystal Structures of a- a n d 0-Funaltrexamine a-FNA
B-FNA
F i g u r e 4. Potential energy vs. C5-C6-N6-C21 torsion angle in a-FNA (left) and P-FNA (right). T h e conformations observed in the X-ray structures are denoted by single arrows. The double arrow points to the torsion angle in a-FNA required t o bring the fumaramate side chain into approximately the same orientation as observed in P-FNA.
in a twist-boat conformation) means that the main point of flexibility for motion of the fumaramate group is rotation about the C(6)-N(6) bond. We have calculated the conformational energy profiles for rotation about the C(6)-N(6) bond in both a- and @-FNAusing a version of the molecular mechanics program CAMSEQ,25 which has been modified to be used in conjunction with the NIH PROPHET computer system.26 CAMSEQ uses empirical potential functions for steric, electrostatic, and torsional interactions, as well as a molecule-solvent term and a molecular dipole-solvent term to evaluate relative energies associated with molecular geometry. The initial conformations for the calculations were obtained from the crystallographic coordinates of aand @-FNA.The relative orientation of the fumaramate group with respect to the morphine fused ring system was varied by rotation about the C(6)-N(6) bond in 10' increments. The energy calculated at each increment was plotted vs. the torsion angle C(5)-C(6)-N(6)-C(21), and the resulting diagram was shifted to place the minimum energy at the zero energy level. The energy profiles are shown in Figure 4. In each instance, the observed solid-state conformation, denoted by arrows in Figure 4, lies in approximately the Weintraub, H.; Hopfinger, A. Znt. J. Quantum Chem., Quantum Biol. Symp. 1975, 2, 203. This data is stored on PROPHET, which is an NIH-sponsored computer network. Information about PROPHET may be obtained from the Director, Chemical/Biological InformationHandling Program, Division of Research Resources, NIH, Bethesda, MD 20205.
J o u r n a l of Medicinal Chemistry, 1986, Vol. 29, No. 5 783
center of a very flat well covering about 120' of conformational space with a very high barrier to rotation outside that region. Using PROPHET^^ we have looked at stereo drawings of the a- and @-FNAstructures at the limits of their allowed conformations; twisting about C(6)-N(6) from -60' to +60° from the observed conformation of @-FNAand -40' to +80° from the observed conformation of a-FNA, that is, through the low-energy regions shown in Figure 4. Examination of these drawings shows that the regions of low energy for each structure are still approximately orthogonal to one another; throughout the 120' rotation the amide carbonyl and ester end of the double bond remain on the same side of the opiate T-frame, the 14-OH side in the case of a-FNA and the phenol 3-OH side in the case of @-FNA(see Figure 2). When the fumaramate group in a-FNA is rotated into the same orientation as in 0-FNA, it is found to be in a region of high potential energy. It suggests that a-FNA cannot easily assume the proposed conformation for the second recognition step to take place and further supports the contention that the general direction of nucleophilic attack is as we have proposed (shown in Figure 3).
Summary and Conclusions The determination of the X-ray crystal structures of aand @-FNA(la, lb) and the conformational energy calculations of the fumaramate moiety in these epimers have revealed a difference in preferred orientation of the electrophilic group. In this regard the T systems of the fumaramate moieties in the epimers bear an orthogonal relation to one another. The results of these studies are consistent with the reportg that only @-FNA(lb) selectively and irreversibly blocks p opioid receptors even though both epimers ( l a , lb) bind reversibly. This supports our earlier proposal that the effectiveness of covalent addition of a receptor-based nucleophile to the reversibly bound ligand is dependent upon the direction of nucleophilic Thus the lack of reactivity of a-FNA toward p receptors is attributed to improper alignment (see Figure 3) between the nucelophile and the Michael acceptor group. It is this second recognition process that is responsible for the recognition amplification observed with @-FNA. Acknowledgment. This work was supported in part by Grant DA-05133 (P.S.P.)from the National Institute on Drug Abuse.
